Journal of Marine Science and Engineering

Article Anatomy and Ultrastructure of the Cyprid Temporary Adhesive System in Two Species of Acorn

Joshua J. Raine 1,*, Nick Aldred 1,2 and Anthony S. Clare 1

1 School of Natural and Environmental Sciences, Newcastle University, Newcastle upon Tyne, Newcastle NE1 7RU, UK; [email protected] (N.A.); [email protected] (A.S.C.) 2 School of Life Sciences, University of Essex, Colchester CO4 3SQ, UK * Correspondence: [email protected]

 Received: 30 October 2020; Accepted: 24 November 2020; Published: 27 November 2020 

Abstract: Acorn are sessile as adults and select their settlement site as a cypris larva. Cyprids are well adapted to exploring surfaces in dynamic environments, using a temporary adhesive secreted from the antennules to adhere during this process. The temporary adhesive and the secretory structures are poorly characterized. This study used serial block-face scanning electron microscopy and three-dimensional modelling to elucidate the anatomy related to temporary adhesion. The temporary adhesive glands of two acorn barnacle species, Balanus amphitrite and , were located in the proximal region of the first antennular segment, contrary to previous descriptions that placed them in the more distal second segment. The temporary adhesive systems of these acorn barnacles are therefore similar to that described for the stalked barnacle, Octolasmis angulata, although not identical. Knowledge of the true location of the temporary adhesive glands will underpin future studies of the production, composition and secretion of the adhesive.

Keywords: barnacles; larvae; anatomy; adhesion; electron microscopy

1. Introduction Barnacle cypris larvae use a proteinaceous adhesive secreted from their paired antennules to temporarily attach to the substratum, allowing them to effectively explore dynamic environments [1,2]. This adhesive is secreted via pores on the third antennular segment and contains the settlement-inducing protein complex (SIPC), or an analogue [3]. Temporarily adhered cyprids detach via mechanical forces, twisting their antennule and leaving behind a “footprint” of adhesive material [2,4–7]. Neither the temporary adhesive nor the structures related to its synthesis and secretion have been fully characterised in acorn barnacles. The temporary adhesive system is distinct from the permanent adhesive used by the cyprid at the point of fixation. The morphology of the cyprid permanent adhesive system has been well characterised [8–10], although only basic compositional data have been obtained. The permanent adhesive is produced and stored in a pair of glands situated behind the compound eyes, with each gland serving a single antennule. When secreted, the cement is transported to the attachment disc by a single duct, which radiates at the surface to numerous pores, spreading it over a wider area. Details regarding the composition and morphology of the temporary adhesive system are equally scant. The first description of the temporary adhesive system and its associated structures was provided by Nott and Foster [11] for Semibalanus balanoides; a boreo-arctic, intertidal, acorn barnacle (Cirripedia, Balanomorpha). They noted that the distal region of the second antennular segment contained numerous unicellular glands, arranged around the circumference of the appendage, and postulated that these may be responsible for producing the temporary adhesive [11].

J. Mar. Sci. Eng. 2020, 8, 968; doi:10.3390/jmse8120968 www.mdpi.com/journal/jmse J. Mar. Sci. Eng. 2020, 8, 968 2 of 13 J. Mar. Sci. Eng. 2020, 8, x 2 of 12

TheseThese putativeputative glandsglands extended extended into into ducts ducts that that fed into fedpores into onpores the surfaceon the ofsurface the third of antennularthe third antennularsegment (the segment attachment (the disc,attachment Figure1 ).disc, Supporting Figure 1). rod Supporting structures androd astructures sheath cell and encapsulated a sheath cell the encapsulatedducts in places, the while ducts the in interior places, of while the gland the interior contained of thethe adhesivegland contained material packedthe adhesive within material vesicles, packedand numerous within microtubulesvesicles, and orientednumerous longitudinally microtubules (Figure oriented1). However,longitudinally these (Figure putative 1). glands However, were theseonly 1–2putativeµm in glands diameter were and only did 1–2 not µm appear in diameter to contain and any did of not the appear cellular to structures contain any related of the to cellular protein structuressynthesis orrelated vesicular to protein packaging. synthesis or vesicular packaging.

FigureFigure 1. 1. SchematicSchematic diagram diagram of of the the temporary temporary adhesive adhesive system system located located in in the the distal distal second second antennular antennular segmentsegment as as proposed proposed by by Nott Nott and and Foster [11]. [11]. Not Not to to scale. scale. The The region region detailed detailed in in the the schematic schematic is is highlightedhighlighted in in red red on the inset. A2 A2 = secondsecond antennular antennular segment, segment, A3 A3 = thirdthird antennular antennular segment segment (attachment(attachment disc), disc), CD CD == cementcement duct, duct, TAV TAV = temporarytemporary adhesive adhesive vesicle, vesicle, TAD TAD = temporarytemporary adhesive adhesive duct,duct, SR SR == supportingsupporting rod, rod, MTb MTb == microtubules,microtubules, SP SP == secretorysecretory pores, pores, SC SC = sheathsheath cell, cell, MV MV == microvilli.microvilli.

AA more more recent recent study study on on the the epibiotic epibiotic stalked stalked barnacle barnacle OctolasmisOctolasmis angulata angulata [12][12] provided provided evidence evidence ofof different different anatomy. anatomy. While While many many aspects aspects of of the the des descriptionscriptions of of the the two two species species were were similar, similar, a a major major differencedifference was was that that the the temporary temporary adhesive adhesive glands glands of of O.O. angulata angulata werewere located located in in the the anterior anterior mantle mantle of the cyprid behind the compound eyes, rather than in the antennules. The glands (~20 25 µm) of the cyprid behind the compound eyes, rather than in the antennules. The glands (~20 × 25µm) comprisedcomprised large,large, multicellular multicellular secretory secretory cells, cells, which which contained contained vesicles. vesicles. Ducts fromDucts the from glands the extended glands extendeddown the down length the of thelength antennules, of the antennules, through which through the putative which adhesive-containingthe putative adhesive-containing vesicles were vesiclestransported were to transported the surface to of the each surf attachmentace of each disc attachment [12]. disc [12]. AsAs proposed by YapYap etet al. al. [ 12[12]] it it is is conceivable conceivabl thate that the the “glands” “glands” described described by Nottby Nott and and Foster Foster [11] [11]for S.for balanoides S. balanoidescorresponded corresponded to the to unicellularthe unicellular gland gland extensions extensions observed observed in the in distalthe distal second second and andthird third antennular antennular segments segments of O. of angulata O. angulata. There. There is, therefore, is, therefore, the possibility the possibility that the that anatomies the anatomies of the ofadhesive the adhesive systems systems of the of two the species two species are comparable. are compar Certaintyable. Certainty over the over location the location of the adhesiveof the adhesive glands glandsin acorn in barnacles acorn isbarnacles essential ifis molecularessential (e.g.,if molecular in situ hybridisation) (e.g., in andsitu immunohistochemicalhybridisation) and immunohistochemicaltechniques are to be successfully techniques applied are to be in successfully studies of adhesive applied composition.in studies of adhesive composition. To address this question, the present study used serial block face-scanning electron microscopy (SBF-SEM) to obtain a comprehensive series of images of the antennule, and to track the temporary

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To address this question, the present study used serial block face-scanning electron microscopy (SBF-SEM) to obtain a comprehensive series of images of the antennule, and to track the temporary adhesive system on its route from the disc surface back to to the glands. In addition, the image series allowed for three-dimensional reconstruction and modelling of the cyprid anatomy in the Microscopy Image Browser (MIB) [13]. Using these techniques, this study explored the temporary adhesive system of two acorn barnacle species, Balanus amphitrite (= Amphibalanus amphitrite) and Megabalanus coccopoma, in order to provide a basis for future investigations into the composition and secretion of the adhesive material.

2. Materials and Methods

2.1. Organism Acquisition The cypris larvae of two species, Balanus amphitrite and Megabalanus coccopoma, were prepared for SBF-SEM. Larval production required the culturing of adult populations. Adult broodstock of B. amphitrite was obtained from Duke University Marine Laboratory (DUML), Beaufort, North Carolina (USA). Adults were maintained in ~33 ppt artificial seawater (ASW, Tropic Marin) at 28 ◦C with aeration. Barnacles were fed daily with freshly hatched Artemia sp. (Varicon Aqua Solutions Ltd., Worcester, UK) nauplii supplemented with the diatoms Tetraselmis suecica and Isochrysis galbana algae at weekends. The water was also changed, and the barnacles cleaned daily. Hatching and release of B. amphitrite nauplii were stimulated by removing the adults from water overnight and reintroducing water the following morning. They were kept in the dark, with a single source of illumination to attract the released nauplii. The nauplii were periodically collected by pipette and pooled in beakers containing T. suecica. When their number was sufficient, the nauplii were added to aerated buckets of artificial seawater at a density of no greater than 1000 larvae per litre and cultured at 28 ◦C on a 16 h:8 h L:D cycle. The nauplii were fed 50 mL per liter of culture of T. suecica (~1.3 106 cells mL 1) and I. galbana (5 106 cells mL 1) in a 3:1 ratio initially and after three days. × − × − The nauplii developed to the cyprid stage in five to six days under these conditions. Cyprids were collected from cultures by filtration with a 250 µm mesh, then transferred to dishes of ASW for storage at 4 ◦C until required. Adult broodstock of M. coccopoma was also acquired from DUML and maintained as described for B. amphitrite. For the collection of larvae, buckets containing the adult M. coccopoma were checked each morning for the presence of nauplii. Nauplii were collected and cultured in the same manner as B. amphitrite. The nauplii developed to the cyprid stage in seven to eight days under these conditions. The larvae were filtered from the cultures and returned to clean buckets with fresh ASW and algal feed after four or five days. Cyprids were extracted from cultures by filtration with a 250 µm mesh. Cyprids retained on the mesh were transferred to dishes containing ASW and stored at 4 ◦C until required.

2.2. Sample Preparation Cyprids of each species, (B. amphitrite, n = 6; M. coccopoma, n = 2) were fixed overnight in 2% glutaraldehyde (Agar Scientific) in 0.1 M sodium cacodylate buffer. Once fixed, the samples were processed using a heavy metal staining protocol adapted from Deerinck et al. [14]. Samples were incubated in a series of heavy metal solutions, rinsing three times for five minutes each in distilled water between steps: 3% potassium ferrocyanide in 2% osmium tetroxide for one hour, 10% thiocarbohydrazide for 20 min, 2% osmium tetroxide for 30 min, 1% uranyl acetate overnight at 4 ◦C, and finally lead aspartate solution (0.02 M lead nitrate in 0.03 M in aspartic acid, pH 5.5) for 30 min at 60 ◦C. The samples were then dehydrated through a graded series of acetone to 100% and then impregnated with increasing concentrations of Taab 812 hard resin, with three changes of 100% resin. The samples were embedded in 100% resin and left to polymerise at 60 ◦C for a minimum of 36 h. J. Mar. Sci. Eng. 2020, 8, 968 4 of 13

The resin blocks were trimmed to approximately 0.75 mm by 0.5 mm and glued onto an aluminium pin. To reduce sample charging within the SEM, the block was painted with silver glue and sputter-coated with a 5 nm layer of gold using a Polaron SEM coating unit.

2.3. Serial Block Face-Scanning Electron Microscopy The pin was placed into a Zeiss Sigma SEM incorporating the Gatan 3view system with DMGMS3 software, which allows sectioning of the block in situ and the collection of a series of images in the z-direction. Multiple regions of interest were imaged at variable magnification, pixel scan, section thickness and pixel resolution depending on the species (Table1).

Table 1. Details on the parameters selected for image capture using the Gatan 3view system. “Series” refers to the z-stack of images collected and compiled for three-dimensional model reconstruction, while “Stills” are the images collected for two-dimensional presentation.

Species Magnification Pixel Time Section Thickness Pixel Resolution Series: Series: 20 nm, 1290–1880 Series: 20 µs Series: 100 nm Balanus amphitrite × 2000 2000–3500 3500 Stills: 898–4000 Stills: 20 µs Stills: 100 nm × × × Stills: 10 nm 4000 4000 as required × Series: 25 nm, Series: 646 Megabalanus × Series: 20 µs Series: 100 nm 3000 3000 Stills: 157–6700 × coccopoma × Stills: 20 µs Stills: 100 nm Stills: 5.6 nm–478.5 nm, as required 2000 2000 ×

In the resulting z-stacks, the areas of interest were identified and segmented manually using Microscopy Image Browser (MIB, University of Helsinki) [13]. The segmentations were imported into Amira (FEI) for the construction of the 3-D models. The B. amphitrite antennule and M. coccopoma third antennular segment overlay models used 3090 and 114 sections, respectively.

3. Results

3.1. Balanus amphitrite Cyprids of B. amphitrite were serially sectioned in the transverse plane. This orientation allowed for the best opportunity to identify the temporary adhesive ducts and track them through the antennule from the gland to the disc. However, the orientation of mounting and curvature of the antennules resulted in some sections being taken at an oblique angle. The temporary adhesive gland (one of a pair) was located in the proximal region of the first antennular segment and can be observed in Figure2a,b. The gland was formed of a cluster of 10–15 cells. The gland and component cells were roughly spherocylindrical in shape. The gland was ~40 µm, by 10 µm overall, while individual cells were 8–12 µm by 4–5 µm. Therefore only a few cells sat in parallel. A summary of the dimensions of key structures is presented in Table2. The components for protein production and packaging were present in the gland cells (Figure2a,b). Rough endoplasmic reticulum and Golgi bodies were clearly visible, along with mitochondria. In a number of these cells, newly formed vesicles (50–200 nm) could be observed prior to their transportation down the antennule (Figure2a,b). While the contents of the vesicles were homogenous in appearance, their size was more variable compared to those observed distally in the first and second antennular segments (Figure2c,d). Much of the proximal first antennular segment was filled with muscle tissue, in various orientations, and the temporary adhesive gland was surrounded by muscle. The cement duct was also evident in this region and was distinct from the temporary adhesive gland as it proceeded proximally into the cephalon where the cement gland was located (Figure2a,b). Finally, the bundled axons comprising the antennular nerve were observed, which would eventually connect with the antennular soma cluster and brain if followed proximally (Figure2b). J. Mar. Sci. Eng. 2020, 8, 968 5 of 13

Table 2. Summary of the dimensions of the cyprid species imaged, and the key structures of their temporary adhesive systems ( standard deviation). ± TAV = temporary adhesive vesicle, TAD = temporary adhesive duct, TAG = temporary adhesive gland, A1 = first antennular segment, A2, second antennular segment, A3 = third antennular segment.

Species TAV Diameter TAD Diameter TAG Cells TAG Cyprid Length A1 Length A2 Length A3 Diameter Balanus amphitrite ~250 nm ( 89) ~0.5 µm ( 0.11) ~4.5 4.5 10 µm ~10 10 40 µm ~400 µm ~90 µm ~80 µm ~20 µm ± ± × × × × Megabalanus coccopoma ~400 nm ( 104) ~0.8 µm ( 0.26) ~5 5 25 µm ~25 25 50 µm ~500 µm ~150 µm ~140 µm ~35 µm ± ± × × × × J. Mar. Sci. Eng. 2020, 8, 968 6 of 13 J. Mar. Sci. Eng. 2020, 8, x 5 of 12

Figure 2. SBF-SEM images of a Balanus amphitrite cyprid temporary adhesive gland and ducts. The Figure 2. SBF-SEM images of a Balanus amphitrite cyprid temporary adhesive gland and ducts. region where the image was captured is highlighted in red on the schematic inset: (a,b) = proximal The region where the image was captured is highlighted in red on the schematic inset: (a,b) = proximal first antennular segment, (c) = proximal second antennular segment, (d) = distal second antennular first antennular segment, (c) = proximal second antennular segment, (d) = distal second antennular segment. TAG = temporary adhesive gland, TAV = temporary adhesive vesicle, TAD = temporary segment. TAG = temporary adhesive gland, TAV = temporary adhesive vesicle, TAD = temporary adhesive duct, N = nucleus, MI = mitochondria, G = Golgi body, AX = axons, CM = cell membrane, adhesive duct, N = nucleus, MI = mitochondria, G = Golgi body, AX = axons, CM = cell membrane, RER = rough endoplasmic reticulum, M = muscle, CD = cement duct, Cu = cuticle. RER = rough endoplasmic reticulum, M = muscle, CD = cement duct, Cu = cuticle.

TheProceeding components distally for protein down theproduction first antennular and packaging segment, were the present temporary in the adhesive gland cells gland (Figure cells 2a,b).transitioned Rough endoplasmic into narrow ducts. reticulum The morphologyand Golgi bodies of the were temporary clearly adhesivevisible, along ducts with remained mitochondria. relatively Inconstant a number through of these the firstcells, and newly second formed antennular vesicles segments (50–200 nm) (Figure could2c,d). be Theobserved temporary prior adhesive to their transportationducts did occasionally down the vary antennule in width, (Figure however, 2a,b). depending While the oncontents the number of the ofvesicles vesicles were contained homogenous within. inFor appearance, the individual their illustrated, size was themore ducts variable contained comp fewared vesicles to those for observed much of theirdistally length in andthe first remained and secondnarrow antennular as a result segments (Figure2c,d). (Figure The 2c,d). cement Much duct of remained the proximal similar first in antennular size but became segment flattened was filled as it withapproached muscle tissue, the junction in various of the orientations, second and thirdand the antennular temporary segments adhesive (Figure gland2c,d). was The surrounded axons of theby muscle.antennular The nervecement followed duct was a similar also pathevident through in th theseis region segments, and was remaining distinct clustered from the (Figure temporary2d). adhesiveThe gland ducts as then it proceeded entered the proximally third antennular into th segment,e cephalon or where attachment the cement disc. Heregland the was temporary located (Figureadhesive 2a,b). ducts Finally, expanded the bundled noticeably, axons becoming comprising larger the spherocylindrical antennular nerve structures were observed, ~1–1.5 whichµm in woulddiameter eventually with two connect distinct with regions the (Figureantennular3). The soma inner cluster region and seemed brain if to followed be the transporting proximally duct (Figure and 2b).contained numerous homogenous vesicles, each ~200 nm in diameter. These vesicles were surrounded by numerousProceeding peripheral distally microtubulesdown the first (Figure antennular3b,c). The segment, outer section the temporary was identifiable adhesive by thegland presence cells transitioned into narrow ducts. The morphology of the temporary adhesive ducts remained relatively constant through the first and second antennular segments (Figure 2c,d). The temporary adhesive ducts did occasionally vary in width, however, depending on the number of vesicles contained

J. Mar. Sci. Eng. 2020, 8, x 6 of 12 within. For the individual illustrated, the ducts contained few vesicles for much of their length and remained narrow as a result (Figure 2c,d). The cement duct remained similar in size but became flattened as it approached the junction of the second and third antennular segments (Figure 2c,d). The axons of the antennular nerve followed a similar path through these segments, remaining clustered (Figure 2d). The ducts then entered the third antennular segment, or attachment disc. Here the temporary adhesive ducts expanded noticeably, becoming larger spherocylindrical structures ~1–1.5 µm in diameter with two distinct regions (Figure 3). The inner region seemed to be the transporting duct J. Mar. Sci. Eng. 2020 8 and contained numerous, , 968 homogenous vesicles, each ~200 nm in diameter. These vesicles were7 of 13 surrounded by numerous peripheral microtubules (Figure 3b,c). The outer section was identifiable byof the supporting presence rodsof supporting (Figure3b,c). rods The (Figure ducts 3b,c). and associatedThe ducts elementsand associated did not elements appear todid contain not appear any of tothe contain cellular any components of the cellular required components to produce requir anded to package produce the and temporary package the adhesive. temporary The adhesive. number of Theducts number correlated of ducts with correlated the number with of the cells number in the temporaryof cells in the adhesive temporary gland. adhesive Towards gland. the disc Towards surface, thesecretory disc surface, ducts secretory lost the ducts supporting lost the outer supportin sectiong outer (Figure section3a) and (Figure the secretory3a) and the pores secretory for both pores the fortemporary both the temporary adhesive and adhesive cement and duct cement could beduct seen could terminating be seen terminating among the microvilli among the that microvilli cover the thatsurface cover of the the surface attachment of the disc attachment (Figure3 disca). (Figure 3a).

FigureFigure 3. 3.SBF-SEMSBF-SEM image image ofof aaBalanus Balanus amphitrite amphitritecyprid: cyprid: (a) = (thirda) = antennularthird antennular segment, segment, (b,c) = temporary (b,c) = temporaryadhesive adhesive ducts within ducts the within third antennularthe third antennular segment. segment. The region The where region the where image the was image captured was is capturedhighlighted is highlighted in red on the in schematic red on the inset. schematic TAD = temporary inset. TAD adhesive = temporary duct, TAV adhesive= temporary duct, TAV adhesive = temporaryvesicle, SP adhesive= secretory vesicle, pore, SP = CD secretory= cement pore, duct, CD NR= cement= neurone, duct, NR AX == neurone,axon, MT AX= = microtubules,axon, MT = microtubules,SR = supporting SR = rods,supporting MV = microvilli.rods, MV = microvilli.

TheThe cement cement duct duct remained remained flattened flattened as as it itexten extendedded into into the the third third antennular antennular segment. segment. As As the the specimenspecimen in inFigure Figure 11 had had not not begun begun the the process process of of permanent permanent adhesion, adhesion, the the duct duct was was empty empty and and appearedappeared to tohave have narrowed narrowed to toa aslit slit of of ~3 ~3 x x< <0.50.5 µm.µm. Presumably, Presumably, this this slit slit would would stretch stretch and and open open duringduring secretion secretion of ofthe the permanent permanent cement cement (Figure (Figure 3a3a).). At At the the disc disc surface, surface, this this singular singular permanent permanent cementcement duct duct diverged diverged into into many many smaller smaller channels channels to to dispense dispense th thee adhesive adhesive over over a alarger larger area. area. Finally,Finally, the the axons axons comprising comprising the the antennular antennular nervenerve connectedconnected toto neuronesneurones serving serving sensilla sensilla on on the thethird third and and fourth fourth antennular antennular segments segments (Figure (Figure3a). 3a). TheThe two-dimensional two-dimensional images images (Figures (Figures 2 2and and 3)3) allowed allowed for for detailed detailed ultrastructural ultrastructural observation observation ofof sections sections of ofthe the cyprid cyprid adhesive adhesive systems. systems. Howe However,ver, the the morphology morphology of ofthe the complete complete system system is isnot not easyeasy to to visualisevisualise from from two-dimensional two-dimensional sections sections alone. alone. To this end,To this a three-dimensional end, a three-dimensional reconstruction reconstructionof the systems of described the systems for describedB. amphitrite forwas B. amphitrite produced was (Figure produced4). Here (Figure the division 4). Here of the the division singular ofcement the singular duct intocement the duct smaller into channels the smaller across channels the disc across surface the was disc visible, surface as was was visible, the position as was ofthe the cement gland behind the eye. Also modelled were two of the 10–15 temporary adhesive ducts, which led back to the temporary adhesive gland in the proximal first segment, where the widening and elongation of the cells could be seen. J. Mar. Sci. Eng. 2020, 8, x 7 of 12 position of the cement gland behind the eye. Also modelled were two of the 10–15 temporary adhesive ducts, which led back to the temporary adhesive gland in the proximal first segment, where theJ. Mar. widening Sci. Eng. and2020, elongation8, 968 of the cells could be seen. 8 of 13

FigureFigure 4. 4. 3-D3-D models models of of B. amphitrite antennuleantennuleand and adhesive adhesive systems, systems, reconstructed reconstructed from from an SBF-SEMan SBF- SEMseries. series. (a) = (laterala) = lateral view, view, (b) = dorsal(b) = dorsal view. Theview. antennular The antennular segments segments were left were open left toallow open viewingto allow of viewingthe interior. of the Lilac interior.= cement Lilac duct = cement/gland, duct/gland, green = temporary green = temporary adhesive duct adhesive/gland cell,duct/gland black = cell,compound black = eye,compound blue = 1st eye, antennular blue = 1st segment, antennular grey =segment,2nd antennular grey = segment,2nd antennular yellow = segment,3rd antennular yellow segment, = 3rd antennularpurple = 4th segment, antennular purple segment. = 4th antennular segment.

3.2. Megabalanus coccopoma 3.2. Megabalanus coccopoma B. amphitrite M. coccopoma ItIt seemed seemed likely likely that that the the adhesive adhesive systems systems of of B. amphitrite andand M. coccopoma wouldwould be be similar, similar, howeverhowever confirmation confirmation of of the the new new observations observations in in a asecond second acorn acorn barnacle barnacle species species was was nevertheless nevertheless B. amphitrite important.important. Since Since thethe temporarytemporary adhesive adhesive gland gland of of B. amphitritehad already had already been located, been thelocated, sectioning the sectioningof M. coccopoma of M.cyprids coccopoma was cyprids changed was from changed transverse from to longitudinaltransverse to to observelongitudinal the structures to observe from the an structuresalternate angle.from an alternate angle. TheThe initial initial longitudinal longitudinal sections sections revealed revealed the the core core structures, structures, namely namely the the antennule, antennule, the the brain brain andand oil oil cells cells within within the the anterior anterior cephalon, cephalon, and and the the thoracic thoracic appendages appendages within within the the thorax thorax (Figure (Figure 5a).5a). TheThe temporary temporary adhesive adhesive gland gland was was not not visible visible in in the the initial initial imaging, imaging, however, however, deeper deeper sectioning sectioning revealedrevealed its its location location to to be be consistent consistent with with that that of of B.B. amphitrite amphitrite, ,in in the the proximal proximal region region of of the the first first antennularantennular segment segment (Figure (Figure 5a,b).5a,b). The temporary adhesive gland was approximately 25 50 µm, roughly prolate spheroid in The temporary adhesive gland was approximately 25 × 50 µm,× roughly prolate spheroid in shape andshape comprised and comprised of ~12 ofelongated ~12 elongated cells cellssurrounded surrounded by muscle by muscle (Figure (Figure 5b,c).5b,c). A Asummary summary of of the the dimensionsdimensions of of key key structures structures is ispresented presented in inTable Table 2.2 A. Acloser closer view view of ofthe the gland gland revealed revealed the the cells cells to beto full be fullof homogenous of homogenous vesicles vesicles of 300–400 of 300–400 nm in nm diameter in diameter (Figure (Figure 5c,d).5 Thesec,d). These cells were cells up were to 5 up µm to µ µ wide5 m depending wide depending on vesicular on vesicular content content and and20–25 20–25 µm long.m long. Additionally, Additionally, the the gland gland cells cells contained contained nuclei,nuclei, rough rough endoplasmic reticulumreticulum and and Golgi Golgi apparatus; apparatus; the cellularthe cellular structures structures necessary necessary for protein for proteinproduction production and vesicular and vesicular packaging packaging (Figure (Figure5d). The 5d). vesicles The vesicles near the near Golgi the apparatus Golgi apparatus were smaller were smallerand sparsely and sparsely distributed, distributed, in addition in addition to being to situated being situated on the righton the of right the images, of the images, furthest furthest from the fromantennule the antennule and transporting and transporting ducts (Figure ducts5 (Figurec,d). 5c,d). As was the case in B. amphitrite, the gland cells of M. coccopoma transitioned into ducts as they proceeded distally, narrowing to ~0.5–1 µm (Figure6a). The duct diameter appeared to be correlated with the abundance of vesicles, with fewer vesicles resulting in a narrower duct. Other than this, the morphology of the ducts remained constant through the first and second antennular segments (Figure6a). J. Mar. Sci. Eng. 2020, 8, 968 9 of 13 J. Mar. Sci. Eng. 2020, 8, x 8 of 12

Figure 5. SBF-SEM images of Megabalanus coccopoma: (a) = cyprid, (b) = cyprid anterior mantle, (c) = Figure 5. SBF-SEM images of Megabalanus coccopoma:(a) = cyprid, (b) = cyprid anterior mantle, temporary adhesive gland, (d) = temporary adhesive gland cells. The region where the image was (c) = temporary adhesive gland, (d) = temporary adhesive gland cells. The region where the image captured is highlighted in red on the schematic inset. C = cephalon, TAG = temporary adhesive gland, was captured is highlighted in red on the schematic inset. C = cephalon, TAG = temporary adhesive TAV = temporary adhesive vesicle, OC = oil cell, A1 = first antennular segment, A2 = second gland, TAV = temporary adhesive vesicle, OC = oil cell, A1 = first antennular segment, A2 = second antennular segment, A3 = third antennular segment (attachment disc), Cu = cuticle, TX = thorax, TA antennular segment, A3 = third antennular segment (attachment disc), Cu = cuticle, TX = thorax, = thoracic appendages, M = muscle, B = brain. CM = cell membrane, N = nucleus, G = Golgi body, RER TA = thoracic appendages, M = muscle, B = brain. CM = cell membrane, N = nucleus, G = Golgi body, = rough endoplasmic reticulum. RER = rough endoplasmic reticulum.

AsOnce was thethe temporarycase in B. amphitrite adhesive,ducts the gland reached cells the of thirdM. coccopoma antennular transitioned segment, theyinto ducts expanded as they into proceededlarger, spherocylindrical distally, narrowing structures to ~0.5–1 on µm the (Figure periphery 6a). The of theduct disc diameter close appeared to the surface to be correlated (Figure6b). withThese the structures abundance were of vesicles, over 1.5 µwithm in fewer diameter vesicles and formedresulting of in two a narrower major sections, duct. Other the vesicle-filled than this, the duct morphologyat the centre, of and the the ducts supporting remained sheath constant around thro theugh outside the first (Figure and6 b).second In addition, antennular these segments structures (Figurenumbered 6a). approximately 12; as for the gland cells (Figure6b,c). The temporary adhesive ducts were filledOnce with the the temporary homogenous adhesive vesicles ducts originally reached observed the third withinantennular the temporary segment, they adhesive expanded gland into cells. larger,However, spherocylindrical no vesicles were structures present on between the periphery the spherocylindrical of the disc close structures to the surface and the (Figure disc surface, 6b). These where structuresthe ducts were narrow over and 1.5 feedµm in into diameter secretory and pores formed (Figure of two6b). major In contrast sections, to the the vesicle-filled peripheral, singular,duct at thetemporary centre, and adhesive the supporting ducts, the sheath cement around duct split the intooutside multiple (Figure sub-channels 6b). In addition, and opened these structures out into the numberedcentre of theapproximately disc (Figure 12;6b). as for the gland cells (Figure 6b,c). The temporary adhesive ducts were filled withReconstructing the homogenous the temporary vesicles adhesiveoriginally ducts observed using within three-dimensional the temporary modelling adhesive reveals gland that cells. the However,“collecting no structures” vesicles were occurred present in parallelbetween pairs the sp andherocylindrical were peripheral structures to the cement and the duct disc (Figure surface,6b). whereUnlike the in ductsB. amphitrite, narrowthe and ducts feed took into asecretory circular route pores immediately (Figure 6b). following In contrast the to putative the peripheral, collecting singular,structures temporary (Figure6 adhesivec) and surrounding ducts, the cement muscle duct tissue. split into multiple sub-channels and opened out into the centre of the disc (Figure 6b). Reconstructing the temporary adhesive ducts using three-dimensional modelling reveals that the “collecting structures” occurred in parallel pairs and were peripheral to the cement duct (Figure 6b). Unlike in B. amphitrite, the ducts took a circular route immediately following the putative collecting structures (Figure 6c) and surrounding muscle tissue.

J. Mar. Sci. Eng. 2020, 8, 968 10 of 13 J. Mar. Sci. Eng. 2020, 8, x 9 of 12

Figure 6. SBF-SEM images of Megabalanus coccopoma: (a) = second antennular segment, (b) = third Figure 6. SBF-SEM images of Megabalanus coccopoma:(a) = second antennular segment, (b) = third antennular segment., (c) = third antennular segment overlaid with a 3-D model of temporary adhesive antennular segment., (c) = third antennular segment overlaid with a 3-D model of temporary adhesive ducts (green). The region where the image was captured is highlighted in red on the schematic inset. ducts (green). The region where the image was captured is highlighted in red on the schematic inset. TAD = temporary adhesive duct, TAV = temporary adhesive vesicle, M = muscle, SP = secretory pore, TAD = temporary adhesive duct, TAV = temporary adhesive vesicle, M = muscle, SP = secretory pore, MV = microvilli, CD = cement duct, Cu = cuticle, NR = neurone. MV = microvilli, CD = cement duct, Cu = cuticle, NR = neurone.

4.4. Discussion Discussion ThisThis study study provides provides new new evidence evidence for for the the locati locationon of of the the temporary temporary adhesive adhesive glands glands in in acorn acorn barnaclebarnacle species species and givesgives greater greater insight insight into into the the temporary temporary adhesive adhesive system. system. Contrary Contrary to the findingsto the findingsof Nott andof Nott Foster and [ 11Foster], the [11], temporary the temporary adhesive adhesive glands of glandsBalanus of amphitriteBalanus amphitriteand Megabalanus and Megabalanus coccopoma coccopomawere not were situated not insituated the second in the antennular second antennular segment, segment, but in the but first in (Figurethe first2 a,b,(Figures Figures 2a,b,4,5 4,b and5b and7). 7).Instead, Instead, the the ultrastructure ultrastructure of of the the acorn acorn barnaclesbarnacles appearedappeared more consistent with with that that of of the the stalked stalked barnaclebarnacle OctolasmisOctolasmis angulata angulata [12],[12], though though there there were were some some key key differences. differences. TheThe temporary temporary adhesive adhesive glands glands of of B.B. amphitrite amphitrite andand M.M. coccopoma coccopoma possessedpossessed similar similar morphology morphology andand location. location. The The glands glands were were located located in the proximalproximal regionregion of of the the first first antennular antennular segment segment (Figure (Figures2a,b 2a,band and Figure 5b).5 b).Each Each gland gland is iscomprised comprised of of ~10–15 ~10–15 elongated elongated cells cells filled filled with with homogenous homogenous vesicles vesicles containingcontaining the the packaged packaged temporary temporary adhesive, adhesive, and and the the organelles organelles needed needed for for protein protein synthesis synthesis and and secretionsecretion (Figures (Figure2 a,b2a,b and and Figure 5c,d).5 c,d).Smaller Smaller vesicles vesicles were were observed observed in inthe the glands glands of of both both species, species, situatedsituated in in the the vicinity vicinity of of Golgi Golgi apparatus. apparatus. These These vesicles vesicles may may have have been been undergoing undergoing the theprocess process of aggregation,of aggregation, coalescence coalescence and condensati and condensationon described described by Yap by et Yapal. [12]. et al. In [M.12 ].coccopoma In M. coccopoma, these smaller, these andsmaller more and sparsely more sparselydistributed distributed vesicles were vesicles loca wereted at located the furthest at the furthestpossible possible point from point the from ducts the leadingducts leading to the toantennule, the antennule, indicating indicating an organisati an organisationon of the of thecells cells within within the the gland gland to toproduce produce and and transporttransport the the adhesive adhesive in in a asingle single direction direction (Figure (Figure 5c,d).5c,d). The The glands glands were were roughly roughly spherocylindrical spherocylindrical oror prolate prolate spheroid spheroid in in shape, shape, with with the the overall overall size size correlated correlated to to the the size size of of the the cyprid cyprid of of each each species species (Table(Table 2).2). However, However, the the glands glands did did not not appear appear to to have have the the morphology morphology of of a a typical typical “exocrine “exocrine gland”, gland”, asas each each cell cell fed fed a a single single duct, duct, and these ductsducts diddid notnot combinecombine or or diverge diverge as as they they extended extended towards towards the thethird third antennular antennular segment segment [15 –[15–18].18]. As As Yap Yap et al. et [ 12al.] [12] described described in the in temporarythe temporary adhesive adhesive system system of the ofstalked the stalked barnacle, barnacle,Octolasmis Octolasmis angulata angulata, the ducts, the ducts that carry that thecarry temporary the temporary adhesive adhesive to the discto the surface disc surfaceare simply are simply extensions extensions of the of gland the gland cells cells themselves. themselves. In both In both cases, cases, the the glands glands were were surrounded surrounded by bymuscle muscle tissue tissue in in various various orientations. orientations. AntennuleAntennule locomotion is likely the the primary primary function function of of these these muscles,muscles, as as described described by by Lagersson Lagersson and and Høeg Høeg [19] [19, ],however, however, contraction contraction of of these these muscles muscles may may apply apply pressurepressure to to the the gland gland and and aid aid in in the the secretion secretion of of the the adhesive adhesive [12]. [12]. Following synthesis and vesicular packaging in the temporary adhesive gland, the vesicles containing the proteinaceous adhesive were transported to the third antennular segment via narrow ducts (Figure2c,d and Figure6a). Like the gland cells, the diameter of the ducts and the abundance of vesicles were correlated to the size of the cyprid species (Table2). These vesicles were homogenous in

J. Mar. Sci. Eng. 2020, 8, 968 11 of 13 electron density and shape, suggesting that unlike the permanent cement, the temporary adhesive is a

J.single Mar. Sci. component, Eng. 2020, 8, orx a mixture of components, secreted as a singular phase. 10 of 12

Figure 7. Updated schematic diagram (Figure 11)) ofof thethe temporarytemporary adhesiveadhesive system,system, withwith thethe glandgland located in the proximal firstfirst antennularantennular segment. Not to scale. The The temporary temporary adhesive adhesive gland contains numerous cells for the synthesis and packing of th thee temporary adhesive and and each each cell cell feeds feeds one one duct. duct. The region detailed in the schematic is highlighted in red on the inset. A1 = firstfirst antennular antennular segment, segment, A2 = secondsecond antennular antennular segment, A3 = thirdthird antennular antennular segment segment (attachment disc), CD = cementcement duct, duct, TAG = temporary adhesive gland, TAC = temporary adhesive adhesive gland cell, TAV == temporary adhesive vesicle, TADTAD= = temporarytemporary adhesiveadhesive duct, duct, CS CS= =putative putative collecting collecting structures, structures, previously previously proposed proposed to beto bethe the temporary temporary adhesive adhesive gland gland by by Nott Nott and and Foster Foster [11 ].[11].

FollowingClose to the synthesis surface ofand the vesicular disc, the packaging ducts widened in the into temporary a spherocylindrical adhesive gland, shape, the gaining vesicles a containingsupportive the outer proteinaceous sheath filled adhesive with rod-like were structurestransported (Figures to the third3a–c andantennular6b). These segment structures via narrow were ducts (Figures 2c,d and 6a). Like the gland cells, the diameter of the ducts and the abundance of vesicles were correlated to the size of the cyprid species (Table 2). These vesicles were homogenous in electron density and shape, suggesting that unlike the permanent cement, the temporary adhesive is a single component, or a mixture of components, secreted as a singular phase.

J. Mar. Sci. Eng. 2020, 8, 968 12 of 13 similar to those previously described by Nott and Foster [11], which were interpreted as unicellular glands and the origin of the temporary adhesive. As their ultrastructural descriptions are in close accord with this study, it is proposed that these duct expansions are “collecting structures” for on-demand secretion. The same interpretation was made by Yap et al. [12] for O. angulata. In addition, these “collecting structures” appeared to be arranged in parallel pairs, peripheral to the central cement duct. The reason for this is unknown (Figures3a and6b). The abundance of these “collecting structures” was consistent with the number of cells observed in the temporary adhesive gland, further indicating that the ducts did not diverge. Finally, between the “collecting structures” and the secretory pores on the disc surface, the ducts lost the outer sheath, contained no vesicles and did not branch (Figures3a and6b). Interestingly, 3-D modelling of the ducts revealed that those of M. coccopoma encircled a region of muscle tissue in the centre of the third antennular segment, while those of B. amphitrite did not, suggesting potential species-specific differences (Figures4a,b and6c). Whether this muscle is simply related to articulation of the third and fourth antennular segments, or functions to aid secretion of the temporary adhesive, is unknown. Although similar in many respects, there are notable differences in the temporary adhesive system in the two acorn barnacle species compared to that of the stalked barnacle O. angulata [12]. Primarily, the glands were observed in the proximal region of the 1st antennular segment in the acorn barnacles, as opposed to behind the eye in O. angulata. Secondly, no merging or diverging of the temporary adhesive ducts was observed in the acorn barnacles, while in the stalked barnacle some was described (Figure4a,b). Finally, Yap et al. [ 12] observed no difference in the content of the glands in relation to cyprid age, implying that production is constitutive throughout the cyprid stage. Cyprids of different ages were not compared here, however, the gland and ducts of the B. amphitrite specimens contained considerably fewer vesicles than the M. coccopoma specimens, implying either a species-specific difference or that unknown factors can influence temporary adhesive production. It is possible that these differences are attributed to the distance in the evolutionary relationships between stalked and acorn barnacles [20,21]. In conclusion, this study has corrected a decades-old description of the location and structure of the temporary adhesive glands in acorn barnacle cyprids. Furthermore, variations were identified between individuals of the acorn barnacles studied here and the prior observations for O. angulata. The importance of these apparent variations will only be determined by further investigation of the ultrastructure of these, and a broader spectrum of barnacle species. Such information would allow for improved future studies of the temporary adhesive systems of barnacle larvae, via both morphology, as some aspects remain unclear still, and molecular biology, as isolation of the gland, will allow for the easier characterisation of the still-unidentified temporary adhesive protein(s).

Author Contributions: Conceptualization, J.J.R., N.A., and A.S.C. Methodology; software; validation.; formal analysis; investigation; resources; data curation; writing—original draft preparation.; visualization; project administration; J.J.R. Writing—review and editing; supervision; funding acquisition, A.S.C. and N.A. All authors have read and agreed to the published version of the manuscript. Funding: Funded in part by US Office of Naval Research award N00014-16-1-3125 (A.S.C. and N.A.). Acknowledgments: Thanks to the Newcastle University Electron Microscopy Research Services team for assisting with imaging. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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